Analysis of in vivo neutrophil infiltration in ear inflammation was performed essentially as described previously(40 (link)). Mice were anaesthetized under isoflurane and approximately 10 μl of 1.5 μM MIP-2 (Peprotech) or PBS was intradermally injected into the pinnae until a blister of 5mm diameter was formed. Pinnae were collected after 4 hours, fixed for 20 min in ice cold 4% paraformaldehyde (PFA), blocked and permeabilised with PBS, 5% BSA, 0.5% Triton X-100 for 3 hours at room temperature and washed three times with PBS. Tissues were stained overnight at 4°C with Ly6G-PE (1A8, Biolegend), CD11b-eFluor660 (M1/70, eBioscience) and VEcadherin-Alexa Fluor 488 (eBioBV13, eBioscience), washed in PBS for 1.5 hours, and compressed between two coverslips for imaging. Neutrophil infiltration was imaged using a 20x multi-immersion objective on a Nikon A1R, with at least 10 fields of view taken per pinna and analysed using ImageJ.
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In Vivo Neutrophil Infiltration Assay
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Alexa fluor 488
Cd11b
Cold
Ear inflammation
Immersion
Isoflurane
Mice
Mip 1
Mip 5
Neutrophil infiltration
Paraformaldehyde
Pinna
Tissues
Triton x 100
Corresponding Organization : Babraham Institute
Other organizations : MRC Laboratory of Molecular Biology
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Variable analysis
independent variables
- Intradermal injection of 1.5 μM MIP-2 (Peprotech) or PBS into the pinnae
- Neutrophil infiltration in the pinnae
- Anaesthesia with isoflurane
- Time point of 4 hours after injection
- Fixation in 4% paraformaldehyde (PFA) for 20 min
- Blocking and permeabilisation with PBS, 5% BSA, 0.5% Triton X-100 for 3 hours
- Staining with Ly6G-PE, CD11b-eFluor660, and VEcadherin-Alexa Fluor 488
- Washing in PBS for 1.5 hours
- Imaging using a 20x multi-immersion objective on a Nikon A1R
- Positive control: Intradermal injection of 1.5 μM MIP-2
- Negative control: Intradermal injection of PBS
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